Relaxin and its receptors (RXPF1 and RXFP2) are found in reproductive and non-reproductive tissues of both males and females, across species. In dogs, both relaxin and receptors are found in reproductive tissues of females and play important roles during pregnancy; however, their presence in male reproductive tissues remains unclear. The goal of this study was to explore the presence of both relaxin and receptors in mature dog spermatozoa. Semen samples of 4 adult (≥4 years old) dogs were harvested from a local breeder and diluted (vol:vol) with a pre-warmed Tris extender solution (pH 6.9). Samples were centrifuged and sperm pellets were resuspended in the Tris extender for motility and velocity analyses using computer-assisted sperm analyzer. Semen with over 70% motility were retained for analyses. A subset of those samples (n = 4 dogs) was subjected to total RNA extraction, followed by RT-PCR to amplify relaxin and RXFP1/2 RNA transcripts, with β-actin used as the housekeeping gene. All PCR amplicons were run on a gel electrophoresis for imaging. Another subset of samples (n = 4 dogs) were used for total protein extraction, followed by immunoblotting using anti-human RXFP1 and RXFP2 antibodies. Finally, the remaining subset of samples (n = 4 dogs) was fixed in 4% paraformaldehyde and submitted to a standard procedure of immunofluorescence using an anti-human relaxin, and followed by fluorescein isothiocyanate-conjugated secondary antibody. Labelled spermatozoa were immediately analysed with flow cytometry and imaged with a confocal microscope. Gene expression data were collected after 40 cycles of PCR and all samples expressed β-actin. The detection of RXFP2 was observed at a low level in all samples, whereas RXFP1 and relaxin were absent. Protein analysis with western immunoblotting showed weak signal bands of both receptors in all samples; however, the band corresponding to RXFP1 (~82 kDa) appeared stronger than that of RXFP2 (~90 kDa). Flow cytometry revealed small proportions of relaxin immunopositive spermatozoa (less than 35%) that exhibited low fluorescence intensity. Confocal microcopy imaging confirmed the weak signals that were mostly located in the sperm head. Altogether, the findings showed that relaxin and its receptors RXFP1 and RXFP2 are present in mature spermatozoa of adult dogs. More studies are needed to better characterise the roles of the relaxin system on canine sperm fertility.